The long term objective of this proposal is to elucidate some of the fundamental mechanisms of the accumulation of amyloid deposits in Alzheimer's disease. We plan to investigate how the structure of the amyloid A4/beta protein, the major protein component of senile plaque amyloid deposits in Alzheimer's brain tissue, is related to the intrinsic biochemical properties of the protein in terms of its solubility properties and ability to assemble into amyloid-like filaments. We also plan to investigate how the biochemical properties of the A4/beta protein are related to the mechanism of amyloid deposition in terms of the proteolytic processing of the amyloid precursor protein and its resistance to degradation by cells. Key amino acid residues will be identified which are necessary for the unique fibril assembly and aggregation properties of the A4/beta protein, by synthesizing A4/beta analogs and characterizing the effects of amino acid replacements on assembly and aggregation. The basis for the differences in the solubility properties of senile plaque amyloid and the properties of the A4/beta protein in vitro will be examined. The role of other components that have ben identified in senile plaque in promoting amyloid fibril formation and stabilizing the aggregated state of the synthetic A4/beta protein will be investigated. We hope to establish some of the molecular details for the aggregation and fibril assembly properties of the peptide by 2-D NMR and chemical crosslinking studies on the wild type and mutant peptides. We will screen for compounds and treatments that interfere with aggregation and fibril formation using a rapid and facile assay. A major focus of this proposal is to determine the mechanism for the intracellular accumulation of the A4/beta protein and its resistance to degradation. The relevance of degradation resistance to the mechanisms of amyloid deposition and the molecular pathogenesis of Alzheimer's disease will be investigated. The structure and aggregation state of the accumulated intracellular fraction of the A4/beta protein will be established. The subcellular localization of the intracellular peptide will be determined. We will determine whether the assembled or aggregated states of the peptide are intrinsically resistant to proteolysis or whether the A4/beta protein escapes degradation by some other mechanism. The hypothesis that the segment of the unprocessed amyloid precursor protein that contains the A4/beta protein is selectively resistant to degradation will be tested to determine whether lysosomal processing of the precursor protein is a plausible mechanism for the production of amyloid. Potential differences in the fate of the intracellular A4/beta protein in normal fibroblasts, fibroblasts from Alzheimer's disease patients and cultured neurons will be explored. The fate of the intracellular peptide and long term effects of intracellular accumulation of the A4/beta protein on cultured cells will be explored.